Original research article

A brief version of this protocol appeared in:

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Abstract

Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.

In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOFTM (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.

96-well plate: 300 μl well volume (250 μl washes) or 1.2 ml well volume (500 μl washes) Note: If using a 300 μl plate, do an additional wash step (resuspension and centrifugation) at each point.

Biosafety cabinet

Centrifuge

Calibrated pipettes

ViCell (Beckman Coulter) or Hemocytometer cell counter

CyTOFTM version 1 mass cytometer (Fluidigm Sciences)

Software

FlowJo software

Microsoft Excel

Procedure

Thaw PBMC

Warm Complete RPMI media to 37 °C in water bath. Each sample will
require 20 ml of media with benzonase. Calculate the amount needed to
thaw all samples, and prepare a separate aliquot of warm media with
1:10,000 benzonase (25 U/ml final concentration; benzonase removes
reduces viscosity and background from free DNA from lysed cells). Thaw
no more than 10 samples at a time.

Remove samples from liquid nitrogen and transport to lab on dry ice.

Place 10 ml of warmed benzonase media into a 15 ml tube, making a separate tube for each sample.

Thaw frozen vials in 37 °C water bath.

When cells are nearly completely thawed, carry to hood.

Add 1 ml of warm benzonase media from appropriately labeled
centrifuge tube slowly to the cells, then transfer the cells to the
centrifuge tube. Rinse vial with more media from centrifuge tube to
retrieve all cells.

Continue with the rest of the samples as quickly as possible.

Centrifuge cells at (RCF = 483) for 10 min at room temperature.

Remove supernatant from the cells and resuspend the pellet by tapping the tube.

Calculate the resuspension volume needed to obtain 1 million viable cells. Note: It is typical to recover 4-8 x 106 cells from a vial frozen at 10 x 106 cells/vial.

Stain cells
Day one

Add 1 million viable cells from a donor into a well of the 96 well
plate. Repeat for all samples. Add CyFACS buffer to approximately 600 μl
and centrifuge cells (RCF = 483) for 10 min at room temperature.

Flick or aspirate to remove supernatant, and repeat wash step and centrifugation with 500 μl CyFACS.

Make cocktail in CyFACS buffer of metal-chelating polymer-labeled
antibodies according to previously determined titration. Make sufficient
volume for each sample to have 50 μl of cocktail. Pipet into 0.1 μm
spin filter and centrifuge in a tabletop microcentrifuge (RCF = 14,000)
for 10 min at room temperature.

Flick or aspirate to remove
supernatant from second wash step B2. Add 50 μl antibody cocktail to
each sample. Pipet up and down to mix. Incubate on ice for 60 min.

Add 100 μl of 2% PFA (in CyPBS); pipet to mix. Place
at 4 °C overnight. PFA fixation is required due to permeabilization and
MilliQ water wash osmotic stress in Day two.

Day two

Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 805) for 10 min
at 4 °C. Flick or aspirate to remove supernatant, resuspend pellet in
500 μl CyFACS, and centrifuge cells (RCF = 805) for 10 min at 4 °C.Note: It is common to increase RCF after fixation, particularly during
permabilization steps. Live cells in Day 1 cannot take the stress.

Make 1x saponin permeabilization buffer in CyPBS. Flick or aspirate
to remove supernatant from step 9. Add 100 μl of 1x saponin
permeabilization buffer to each sample, pipet to mix. Incubate on ice
for 45 min.

Make 1:2,000 dilution in CyPBS
of Ir-intercalator. Add 100 μl of diluted Ir-intercalator solution to
each sample, pipet to mix. Incubate at room temperature for 20 min.

Add 500 μl CyFACS buffer, then centrifuge cells (RCF = 805) for 10
min at room temperature. Flick or aspirate to remove supernatant,
resuspend pellet in 500 μl CyFACS, and centrifuge cells (RCF = 805) for
10 min at 4 °C. Flick or aspirate to remove supernatant, and repeat at
least twice with 500 μl of MilliQ water. The MilliQ water washes are
critical to remove buffer salts, which can cause the Current setting on
the CyTOF to drift over the course of your experiment.

Acquire samples on the CyTOF, after standard instrument setup procedures. Collect at least one “push” of 500 μl per sample.

Analyze data

Analyze data using FlowJo software using the standard CyTOF template.
Adjust gates on a donor-specific basis, if necessary, to control for any
differences in background or positive staining intensity. Export the
statistics for each gated population to an Excel spreadsheet.

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